Nucleotide sequence analysis of endogenous murine leukemia virus-related proviral clones reveals primer-binding sites for glutamine tRNA.

Nucleotide sequences of the region that corresponds to the site of tRNA primer binding for a functional retrovirus were determined in five murine leukemia virus-related sequence clones from mouse chromosomal DNA, which contain a unique 170 to 200-base-pair additional internal segment in the long terminal repeats. The 3'-terminal 18-nucleotide sequence of a major glutamine tRNA isoacceptor was found to match well with the putative primer binding site: 18 of 18 in three clones, 17 of 18 in one clone, and 16 of 18 in one clone. This implies that most of these endogenous proviral sequences of the mouse genome, if replicated as retroviruses, will be different from ecotropic murine leukemia viruses and most mammalian type C retroviruses in using glutamine tRNA, rather than proline tRNA, as a primer.     

Mutated primer binding sites interacting with different tRNAs allow efficient murine leukemia virus replication.

Two Akv murine leukemia virus-based retroviral vectors with primer binding sites matching tRNA(Gln-1) and tRNA(Lys-3) were constructed. The transduction efficiency of these mutated vectors was found to be comparable to that of a vector carrying the wild-type primer binding site matching tRNA(Pro). Polymerase chain reaction amplification and sequence analysis of transduced proviruses confirmed the transfer of vectors with mutated primer binding sites and further showed that tRNA(Gln-2) may act efficiently in conjunction with the tRNA(Gln-1) primer binding site. We conclude that murine leukemia virus can replicate by using various tRNA molecules as primers and propose primer binding site-tRNA primer interactions to be of major importance for tRNA primer selection. However, efficient primer selection does not require perfect Watson-Crick base pairing at all 18 positions of the primer binding site.